Elucidation of Distinct Modular Assemblies of Smoothened Receptor by Bitopic Ligand Measurement

Abstract

Class F G protein-coupled receptors are characterized by a large extracellular domain (ECD) in addition to the common transmembrane domain (TMD) with seven α-helixes. For smoothened receptor (SMO), structural studies revealed dissected ECD and TMD, and their integrated assemblies. However, distinct assemblies were reported under different circumstances. Using an unbiased approach based on four series of cross-conjugated bitopic ligands, we explore the relationship between the active status and receptor assembly. Different activity dependency on the linker length for these bitopic ligands corroborates the various occurrences of SMO assembly. These results reveal a rigid “near” assembly for active SMO, which is in contrast to previous results. Conversely, inactive SMO adopts a free ECD, which would be remotely captured at “far” assembly by cholesterol. Altogether, we propose a mechanism of cholesterol flow-caused SMO activation involving an erection of ECD from far to near assembly.

Designing drugs and chemical probes with the dualsteric approach

“…In addition, FRET also suffers from false-positive fluorescent signals. 69 In the design of dualsteric modulators, an important step is to decide the length of linkers suitable to bridge the orthosteric and allosteric site. In reverse, we could also enumerate linkers with different lengths to measure the possible distance between the two sites.…”

“…The dualsteric approach has also been used aside from making drugs, like the design of highly-sensitive fluorescent tracer 155,221 and molecular rulers 69,133 for unbiased mechanism study. Relying on the good properties of dualsteric binding, other biological applications could also be applied.…”

“…After a manual filtering, 133 research articles focusing on identifications of novel dualsteric modulators were found. Modulators targeting GPCR 50,53,56–137 or kinase 138–157 account for more than three quarters (Fig. 5).…”

“…GPCR itself has accommodated 62.4% targets of reports. 50,53,56–137 GPCR is a class of 7-transmembrane (7TM) proteins decorated on the cell membrane for transduction amplification of exterior signals into cells. 189 They are vital in many physiological processes and are therefore popular drug targets for a series of diseases like cancers and neurological disorders.…”

“…Another example was given by Zhao and coworkers. 69 Smoothened receptor (SMO) is a GPCR composed of an extracellular domain (ECD) and a transmembrane domain (TMD). The orthosteric site is in TMD, while an allosteric site was found in ECD.…”